Herbicidal composition for the control of annual bluegrass comprising xanthomonas campestris and sulfonylurea herbicides

ABSTRACT

The present invention relates to a herbicidal composition for the control of annual bluegrass, comprising a microorganism having an ability to control annual bluegrass and belonging to the genus Xanthomonas, and a sulfonylurea compound. The composition of the present invention shows excellent herbicidal activity against annual bluegrass and broad-leaved weeds, and is extremely useful as a herbicide for turf.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a herbicidal composition for thecontrol of annual bluegrass (Poa annua) comprising a microorganismbelonging to the genus Xanthomonas.

2. Prior Art

Annual bluegrass which is growing abundantly at golf courses, cityparks, athletic grounds and the like is the most strong, harmful weed inthe turf. This weed is widely distributed throughout the world. Inparticular, annual bluegrass mixed in with the turf of golf courses,such as putting greens, tee grounds, fairways and roughs, comes intoears at all times in spite of frequent mowing, and scatters a largequantity of seeds into the turf all the year round. At present, thereare a number of herbicides developed for control of annual bluegrass.

However, the effects of these chemical herbicides are very unstable atthe site of use, and this has led to an increase in the amount of useand the frequency of application of these chemicals. The abundant use ofagricultural chemicals at golf courses has particularly become a bigsocial problem as one of the causes of environmental pollution. Amongthese chemical herbicides, there is no foliar treatment agent which canselectively kill annual bluegrass mixed in with Western turfgrasses,such as bentgrass, without harming the desired turfgrasses. Therefore,for the maintenance of bent green, manual weeding or even total renewalof turf is inevitably required, and the burden of costs therefor istremendous.

On the other hand, microbial herbicides are being developed in theUnited States and Japan which comprise a bacterial pathogen to a plant,Xanthomonas campestris, and which selectively controls annual bluegrasswithout polluting the environment. Furthermore, a mixed application ofXanthomonas campestris and a chemical agent [a plant growth regulator,mefluidide (Embark™)] for enhancing the herbicidal effect of themicrobial herbicide has also been disclosed [Japanese Unexamined PatentPublication (Kohyo) No. 63-502438, U.S. Pat. No. 5,192,541].

However, the method of the prior art wherein the bacterial pathogen,Xanthomonas campestris, and the plant growth regulator are mixed andapplied together has not solved at all a problem of herbicial spectrumwhich is the greatest advantage and, at the same time, a drawback ofmicrobial herbicides, though this process can control annual bluegrasseffectively. In other words, this prior art process can control onlyannual bluegrass.

OBJECTS AND SUMMARY OF THE INVENTION

It is the object of the present invention to solve the problem ofherbicidal spectrum of microorganisms belonging to the genusXanthomonas, and to enhance the ability of those microorganisms tocontrol annual bluegrass.

The present inventors have found that the addition of a sulfonylureacompound exhibiting herbicidal activity against broad-leaved weeds to amicroorganism having an ability to control annual bluegrass andbelonging to the genus Xanthomonas can not only expand the herbicidalspectrum to broad-leaved weeds, but also remarkably improve the abilityof the microorganism to control annual bluegrass. The present inventionhas been accomplished based on this finding.

According to the present invention, there is provided a herbicidalcomposition for the control of annual bluegrass, comprising amicroorganism having an ability to control annual bluegrass andbelonging to the genus Xanthomonas, as well as a sulfonylurea compound.Examples of microorganisms having an ability to control annual bluegrassand belonging to the genus Xanthomonas include microorganisms belongingto the species Xanthomonas campestris. More concretely, Xanthomonascampestris strains P-482, P-484 and the like may be cited. Examples ofsulfonylurea compounds to be used in the present invention includeimazosulfuron, flazasulfuron, pyrazosulfuron-ethyl, benzosulfuron-methyland the like.

DETAILED DESCRIPTION OF THE INVENTION

Now the present invention will be described in more detail.

The microorganism to be used for the present invention is notparticularly limited as long as it belongs to the genus Xanthomonas andhas an ability to control annual bluegrass. Preferably, a microorganismbelonging to the species Xanthomonas campestris is used. Preferablestrains of Xanthomonas campestris include P-482, P-484, P-481, P-485,P-496, P-497, P-498, P-499, P-500, P-515, P-516 and P-517. These strainshave been isolated from the internal parts of plant bodies of naturallygrowing annual bluegrass.

Out of the above strains, P-482 shows the following bacteriologicalproperties.

    ______________________________________                                        Bacteriological properties of P-482                                           Chemical substance                                                                              Decomposing ability                                         ______________________________________                                        α-Cyclodextrin                                                                            +                                                           Dextrin           +                                                           Glycogen          +                                                           Tween 40          +                                                           Tween 80          +                                                           N-acetyl-D-galactosamine                                                                        -                                                           N-acetyl-D-glucosamine                                                                          +                                                           Adonitol          -                                                           L-arabinose       +                                                           D-arabitol        -                                                           Cellobiose        +                                                           i-Erythritol      -                                                           D-fructose        +                                                           L-fucose          +                                                           D-galactose       +                                                           Gentiobiose       +                                                           α-D-glucose +                                                           m-Inositol        -                                                           α-D-lactose +                                                           Lactulose         +                                                           Maltose           +                                                           D-mannitol        -                                                           D-mannose         +                                                           D-melibiose       -                                                           B-methyl-D-glucoside                                                                            -                                                           D-psicose         +                                                           D-raffinose       -                                                           L-rhamnose        -                                                           D-sorbitol        -                                                           Saccharose        -                                                           D-trehalose       +                                                           Turanose          UN                                                          Xylitol           -                                                           Methyl pyruvate   +                                                           Monomethyl succinate                                                                            +                                                           Acetic acid       +                                                           cis-Aconitic acid -                                                           Citric acid       -                                                           Formic acid       -                                                           D-lactone galactonate                                                                           -                                                           D-galactonic acid -                                                           D-gluconic acid   -                                                           D-glucosamic acid -                                                           D-glucuronic acid -                                                           α-Hydroxybutyric acid                                                                     +                                                           β-Hydroxybutyric acid                                                                      -                                                           γ-Hydroxybutyric acid                                                                     -                                                           ρ-Hydroxyphenylacetic acid                                                                  -                                                           Itaconic acid     -                                                           α-Ketobutyric acid                                                                        +                                                           α-Ketoglutaric acid                                                                       +                                                           α-Ketovaleric acid                                                                        -                                                           D, L-lactic acid  +                                                           Malonic acid      -                                                           Propionic acid    -                                                           Quinic acid       -                                                           Sebacic acid      -                                                           Succinic acid     +                                                           Bromosuccinic acid                                                                              +                                                           Succinamide       +                                                           Glucuronamide     -                                                           Alaninamide       +                                                           D-alanine         +                                                           L-alanine         +                                                           L-alanyl-glycine  +                                                           L-asparagine      -                                                           L-aspartic acid   UN                                                          L-glutamic acid   +                                                           Glycyl-L-aspartic acid                                                                          -                                                           Glycyl-L-glutamic acid                                                                          +                                                           L-histidine       -                                                           Hydroxy-L-proline +                                                           L-leucine         -                                                           L-ornithine       -                                                           L-phenylalanine   -                                                           L-proline         -                                                           L-pyroglutamic acid                                                                             -                                                           D-serine          -                                                           L-serine          +                                                           L-threonine       -                                                           D, L-carnitine    -                                                           γ-Aminobutyric acid                                                                       -                                                           Urocanic acid     -                                                           D-saccharic acid  -                                                           Inosine           -                                                           Urdine            -                                                           Thymidine         -                                                           Phenylethylamine  -                                                           Putrescine        -                                                           2-Aminoethanol    -                                                           2,3-Butanediol    -                                                           Glycerol          +                                                           D, L- α-glycerolphosphate                                                                 +                                                           Glucose-l-phosphate                                                                             +                                                           Glucose-6-phosphate                                                                             +                                                           ______________________________________                                         +: can assimilate the substance                                               -: cannot assimilate the substance                                            UN: The results were unstable in repeated tests.                         

The above results were obtained by using Biolog GN MicroPlate™manufactured by Biolog Inc. As a result of a data base survey usingMicrolog™ Software of Biolog Inc. for identifying microorganisms, P-482which has the above-mentioned properties was identified as amicroorganism belonging to the species Xanthomonas campestris. Surveyswere conducted for the other strains according to similar procedures,and all of them were identified as microorganisms belonging to thespecies Xanthomonas campestris. Incidentally, the two strains of P-482and P-484 were deposited with National Institute of Bioscience and HumanTechnology, Agency of Industrial Science and Technology, Tsukuba-shi,Japan, under accession Nos. FERM BP-4431 and FERM BP-4430, respectively,on Oct. 1, 1993.

No special methods are required for the cultivation of the microorganismto be used for the present invention. The microorganism can becultivated according to conventional methods. Either a synthetic mediumor a natural medium may be used as long as it appropriately containsassimilable carbon source and nitrogen source, inorganic substances andnecessary growth promoting substances. For example, YNB (yeast and meatbroth) medium or NA (nutrient agar) medium may be used. During thecultivation, it is preferred that the temperature be kept at 5°-40° C.,preferably 28°-31° C., and pH at 5-9, preferably 6-7. When themicroorganism has been grown for 2 to 4 days under such conditions, asufficient amount of cells can be obtained. In the preparation of thecomposition of the present invention, the use of cells themselves ispreferable. The cells are obtained by centrifugation of cultures of themicroorganism.

The sulfonylurea compound to be used for the present invention is notparticularly limited, as long as the compound has the followingstructure and shows herbicidal effect on broad-leaved weeds:

R₁ --SO₂ --NH--CO--NH--R₂ (wherein R₁ and R₂ are any compounds.)Examples of such sulfonylurea compounds include bensulfuron,chlorimuron, cinosulfuron, flazasulfuron, imazosulfuron, nicosulfuron,primisulfuron, pyrazosulfuron, thifensulfuron, tribehuron, triasulfuron,and methyl or ethyl esters thereof. Among all of these compounds, it ispreferable to use imazosulfuron, flazasulfuron, pyrazosulfuron-ethyl,bensulfuron-methyl and the like. In the composition of the presentinvention, one of these compounds may be used alone, or plurality ofthem may be used in combination. By the way, these sulfonylureacompounds have herbicidal effect upon broad-leaved weeds, but theyscarcely show this effect upon those weeds belonging to the genus Poa,such as annual bluegrass.

When the composition of the present invention is used as a herbicide, itcan be used as a liquid formulation suspended in water. In this case, aspreader or the like may be added, if necessary. If the composition isused as a liquid formulation, the microorganism concentration is 10¹-10¹¹ CFU/ml, preferably 10⁶ -10¹⁰ CFU/ml, and more preferably 10⁷ -10¹⁰CFU/ml. If the concentration is over 10¹¹ CFU/ml, the viscosity of thecomposition increases so much that the use thereof becomes uneasy. Ifthe concentration is below 10³ CFU/ml, the herbicidal effect thereofremarkably decreases. However, if the microorganism of the presentinvetion is used at a low concentration of 10³ CFU/ml or below, itreveals the activity of a plant growth regulator against annualbluegrass. In other words, when applied at a low concentration, themicroorganism of the present invention promotes the growth of culms ofannual bluegrass before seeds thereof attain maturity. This means thatthe seeds of annual bluegrass are removed by mowing before they fallupon the turf. This activity as a plant growth regulator workseffectively even at low temperatures where the herbicidal effect of themicroorganism is relatively suppressed. The concentration of thesulfonylurea compound is 0.001-0.1% by weight, preferably 0.01-0.05% byweight, for imazosulfuron; 0.1-100 ppm, preferably 0.5-50ppm forflazasulfuron; 0.1-50 ppm, preferably 1-10 ppm for pyrazosulfuron-ethyl;and 0.01-0.25% by weight, preferably 0.1-0.25% by weight, forbensulfuron-methyl. In addition, the herbicide of the present inventioncan be used as a dust formulation, not limited to water suspension.

When the composition of the present invention is applied to an actualfield, the composition is used in a manner so that the microorganismconcentration becomes 10¹¹ -10¹⁰ CFU per 10 ares of the field, and theconcentration of sulfonylurea compound 10-100 g per 10 ares of the fieldfor imazosulfuron and 0.025-7.5 g for flazasulfuron.

The target weeds to be controled by the composition of the presentinvention are annual bluegrass, purple nutsedge, dandelion and the like,and the composition of the present invention does not reveal anypathogenicity to major turfgrasses grown at golf courses, such asbentgrass, Kentucky bluegrass, perennial ryegrass, Italian ryegrass,Bermuda grass, tall oatgrass, timothy grass, tall rescue, red rescue,chewing rescue and hard rescue, as well as other gramineous crops.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention will now be described in more detail withreference to the following Test Examples and Examples, which should notbe construed as limiting the scope of the present invention.

(Test Example 1) The isolation, selection and identification ofmicroorganisms

Annual bluegrass (Poa annua) plants growing at golf courses, parks andthe like were collected. After damaged tissues were removed from theplant samples, leaf or stem sections 1 cm in length were prepared. Theywere surface sterilized by dipping into 70% ethanol solution for 1minute and then washing with sterile distilled water. The surfacesterilized sections were milled in 200 μl of sterile distilled water,then streaked onto an ordinary agar medium (NA medium) and incubated inan incubator at 28° C. By the above operations, only those bacteriawhich exist inside of the plant bodies can be separated.

Such colonies that resemble Xanthomonas bacteria were selected from thecolonies appeared on the agar medium during the incubation period. Thethus selected colonies were suspended in a small amount of steriledistilled water. Scissors sterilized with 70% ethanol and flame weredipped in the resultant cell suspension. This suspension was inoculatedinto annual bluegrass seedlings which had been grown for about 1 monthin a greenhouse by cutting the tip of their leaves with theabove-mentioned scissors. About 3 weeks later, Xanthomonas bacteria wereseparated by similar procedures as mentioned above from those annualbluegrass plants whose symptoms of disease had been confirmed. Theseseparated bacteria were inoculated into annual bluegrass plants again,and then the bacterial pathogens of annual bluegrass were separated fromthose plants which were confirmed to exhibit similar symptoms. As aresult, 89 strains of Xanthomonas bacteria showing pathogenicity toannual bluegrass were obtained from 42 locations of 17 urban and ruralprefectures in Japan. From these strains, 12 strains of annual bluegrasspathogens which have extremely strong virulence were screened. They areP-482 (FERM BP-4431), P-484 (FERM BP-4430), P-481, P-485, P-496, P-497,P-498, P-499, P-500, P-515, P-516 and P-517. All of those annualbluegrass plants which had been inoculated with the above 12 strainsbegan wilting from the site of cutting 5 days after the inoculation, and7 days after the inoculation their leaves as a whole wilted whilemaintaining a green color. Their plant bodies as a whole became whiteand died completely 10 days after the inoculation.

(Test Example 2) The pathogenicity of the isolated microorganisms toannual bluegrass

Seeds of annual bluegrass (25 mg) were sown in Jiffy Pots (5 cm x 5 cm x5 cm). The resultant seedlings were grown in a greenhouse for 3 weeks,and then used for inoculation test. Before inoculation, the plants werecut to a height of 2 cm with an electric mower. They werespray-inoculated with 0.5 ml per pot of cell suspensions of Xanthomonasbacteria of which the cell density had been adjusted at 10⁹ CFU/ml. The12 strains of Xanthomonas bacteria [P-482 (FERM BP-4431), P-484 (FERMBP-4430), P-481, P-485, P-496, P-497, P-498, P-499, P-500, P-515, P-516and P-517] used for this test had been respectively grown in advance onYNB (yeast/meat broth) medium at 28° C. for 20 hours. Then, 1 ml each ofthese cultures were transferred to 100 ml of YNB medium, where they werefurther cultivated under shaking at 28° C. After 22 hours, the cultureswere centrifuged at 5000 rpm for 15 minutes to thereby obtain a pellet.The pellet was then diluted with sterile distilled water, and the OD₅₇₅thereof was measured. Inoculation sources of the predetermined celldensity were obtained by calculating 0.1 in absorbance as 1 x 10⁸ODU/ml. Incidentally, 100 μl of the serially diluted inoculation sourcewas spread on an NA (nutrient agar) plate, which was then left in anincubator at 28° C. for 3 days. The accurate viable bacterial count(CFU/ml) was obtained by counting the number of colonies appearing onthe plate. As a control standard, plants were spray-inoculated with theequal amount of distilled water. The inoculated plants were placed in agreenhouse at 25°/20° C. (day temperature/night temperature). After 3weeks from the inoculation, the plants were observed for diseasesymptoms, and the virulence of each strain was compared with each other.

    ______________________________________                                        The pathogenicity of Xanthomonas strains to annual bluegrass                  ______________________________________                                        P-481             +++                                                         P-482             +++                                                         P-484             +++                                                         P-485             +++                                                         P-496             +++                                                         P-497             +++                                                         P-498             +++                                                         P-499             +++                                                         P-500             +++                                                         P-515             +++                                                         P-516             +++                                                         P-517             +++                                                         Control (treated with                                                                           -                                                           sterile distilled water)                                                      ______________________________________                                         +++: Death                                                                    -: No symptoms observed                                                  

As a result, P-482 (FERM BP-4431), P-484 (FERM BP-4430), P-481, P-485,P-496, P-497, P-498, P-499, P-500, P-515, P-516 and P-517 withered andkilled annual bluegrass completely.

(Test Example 3) Assay of the safety of Xanthomonas strains to majorturfgrasses, gramineous plants and useful crops.

Seeds of major turfgrasses, gramineous plants and useful crops were sownin small pots (30 cm x 10 cm x 5 cm) for raising seedlings. Fordicotyledonous plants, those grown up to 3 to 5 true leaves were usedfor the test. For monocotyledonous plants, those grown up to 3 to 5leaves were used for the test. In substantially the same manner asdescribed in Test Example 2, cell suspensions of P-482 and P-484 wereprepared and adjusted to give a cell density of 10⁹ CFU/ml. Sterilizedscissors were dipped into these cell suspensions, and used to cutturfgrass seedlings to a height of 2 cm. For the other plants, bundlesof sterilized needles (7 needles forming a bundle) which had been dippedinto the suspensions were used for inoculation by perforation. The cellsuspension was inoculated into the leaf vein (vascular bundle) of themost developed leaf and 2 other leaves located above it in each plant.Immediately after the inoculation, the plants were carried to a humidchamber with 100% humidity at 25° C. and left there overnight. Then, theplants were placed in a greenhouse at 25° C./20° C. (daytemperature/night temperature). After 2 or 3 weeks, each plant wasobserved for disease symptoms, and the presence of pathogenicity to eachplant was assayed. As control standards, all of the plants were sprayedwith sterile distilled water and a similar assay was conducted. Inaddition, as comparative controls, annual bluegrass plants wereinoculated with P-482 and P-484 by using sterilized scissors andneedles, and the pathogenicity of these strains to annual bluegrass wasconfirmed. For each section, test was repeated 3 times.

The results are shown below.

    ______________________________________                                                              Pathogenicity                                                    Plant          P-482   P-484                                         ______________________________________                                        Major turfgrasses                                                                        Bentgrass        -       -                                                    Kentucky bluegrass                                                                             -       -                                                    Perennial ryegrass                                                                             -       -                                                    Italian ryegrass -       -                                                    Bermuda ryegrass -       -                                                    Tall oatgrass    -       -                                                    Timothy grass    -       -                                                    Tall fescue      -       -                                                    Red fescue       -       -                                                    Chewing fescue   -       -                                                    Hard fescue      -       -                                         Gramineous crops                                                                         Rice             -       -                                                    Barley           -       -                                                    Wheat            -       -                                                    Corn             -       -                                                    Edible millet    -       -                                         Useful crops                                                                             Tobacco          -       -                                         cultivated Pumpkin          -       -                                                    Cucumber         -       -                                                    Carrot           -       -                                                    Burdock          -       -                                                    Spinach          -       -                                                    Green onion      -       -                                                    Onion            -       -                                                    Soy bean         -       -                                                    Garden pea       -       -                                                    Cow pea          -       -                                                    Kidney bean      -       -                                                    Potato           -       -                                                    Sweet potato     -       -                                                    Taro             -       -                                                    Tomato           -       -                                                    Egg plant        -       -                                                    Radish           -       -                                                    Rape             -       -                                                    Cabbage          -       -                                                    Chinese cabbage  -       -                                                    Turnip           -       -                                                    Garland chrysanthemum                                                                          -       -                                                    Lettuce          -       -                                                    Mat rush         -       -                                                    Japanese honewort                                                                              -       -                                                    Violet           -       -                                                    Lily             -       -                                                    Carnation        -       -                                         Control    Annual bluegrass +++     +++                                                  (Needle inoculation)                                                          Annual bluegrass +++     +++                                                  (Scissors inoculation)                                             ______________________________________                                         +++: Death                                                                    -: No symptoms observed                                                  

As a result, it was found that both P-482 and P-484 show nopathogenicity to those plants other than annual bluegrass.

(Test Example 4) The identification of microorganisms

Using a BIOLOG system, bacteriological characteristics were examined forthe 12 strains (P-482, P-484, P-481, P-485, P-496, P-497, P-498, P-499,P-500, P-515, P-516 and P-517 ) which were isolated in Test Example 1.

In the BIOLOG system, there are used Biolog GN MicroPlate™, a computer,and Microlog™ Software which is the data base and software for theidentification of microorganisms. Biolog GN MicroPlate™ is a 96-wellmicrotiter plate containing 96 kinds of chemicals (such as sugars,organic acids, amino acids and the like) to examine the characteristicassimilation of chemicals by microorganisms of interest. Now, theoperational procedures for this system will be described briefly. First,each culture of the above-mentioned 12 strains which have been grown inadvance is placed in all of the wells of Biolog GN MicroPlate™, and thengrown for 1 to 3 days. If the strain has assimilated a chemical, apurple precipitate will be produced (an agent which will produce aprecipitate under certain circumstances is also filled in the well).Then, the results obtained are input to the computer, and search commandis given. Subsequently, the results of identification are automaticallydisplayed.

(Test Example 5) The control effect of Xanthomonas bacteria on annualbluegrass

25 mg of annual bluegrass seeds were sown on the soil filled in a jiffypot 5 cm x 5 cm x 5 cm (out of about 78 seeds, about 50 seedsgerminated). Seedlings were grown in a greenhouse up to 3 to 4 leaves,and used for test during the third week from the sowing. Theabove-mentioned pot was made one section. For each experimental section,the test was repeated 6 times.

In substantially the same manner as described in Test Example 2, P-482and P-484 cultures were adjusted with distilled water to give a celldensity of 10⁹ CFU/ml. Each 3 ml of the thus prepared inoculationsources was spray-inoculated with a sprayer to 6 sections of annualbluegrass plants which had been cut to a height of 2 cm with an electricmower. As a control, sterile distilled water was similarlyspray-inoculated to plants.

After the inoculation, the plants were carried to a greenhouse at 25°C./20° C. (day temperature/night temperature), and observed for thetransition of disease symptoms. Control effect was judged by observingthe appearance of the plants 2 weeks after the inoculation based on thefollowing 6-grade rating. The results are shown as the average valuefrom 6 repeated tests.

0: No influence upon the growth of plants

1: 20% or less of growth suppression by wilt

2: 20-40% of growth suppression by wilt

3: 40-60% of growth suppression by death or wilt

4: 60-80% of growth suppression by death or wilt

5: 80-99% of growth suppression by death or wilt

6: Complete death

    ______________________________________                                                  Control    P-482   P-484                                            ______________________________________                                        Control effect                                                                            0            5.3     5.2                                          ______________________________________                                    

As a result, Xanthomonas strains of P-482 and P-484 pertaining to thepresent invention exhibited excellent herbicidal effect on annualbluegrass.

(Test Example 6) Safety to animals

With respect to the acute dermal toxicity and acute oral toxicity ofP-482 on SD rats, LD₅₀ values are 2000 mg/kg or more and 5000 mg/kg ormore, respectively, for male and female (according to the data measuredby Institute of Environmental Toxicology). Its safety to animals hasbeen proven.

EXAMPLE The Herbicidal Effect of the Herbicidal Composition on AnnualBluegrass

Annual bluegrass seedlings and cells of Xanthomonas strain P-484 wereprepared in substantially the same manner as described in Test Example5. A cell suspension of P-482 was prepared to give a cell density of 2 x10⁸ CFU/ml so that the density becomes 10⁸ CFU/ml when it is mixed witha sulfonylurea compound prepared in a 2-fold concentration. Thesulfonylurea compound to be mixed in the composition was prepared inadvance to give a 2-fold concentration. Imazosulfuron (Shibatait, aproduct of Takeda Chemical Industries Ltd.) was prepared so that itsconcentration becomes 0.05% and 0.01% when applied. Flazasulfuron(Shibagen, a product of Ishihara Sangyo Kaisha Ltd.) was prepared sothat its concentration becomes 50 ppm, 5 pp and 0.5 ppm when applied.

An aliquot of the above-mentioned cell suspension was mixed with theequal amount of each of the sulfonylurea solutions to give a totalvolume of 3 ml. This mixture was spray-inoculated with a sprayer toannual bluegrass seedlings of 6 sections which had been cut to a heightof 2 cm with an electric mower in advance. As a control, steriledistilled water was similarly spray-inoculated to plants.

Since the growth rate of Xanthomonas bacteria vary depending ontemperatures and thus their herbicidal effects vary, the temperature ingreenhouses was set at 3 grades, and the influence thereof was alsostudied. In other words, 3 greenhouses were used wherein the temperature(day temperature/night temperature) was set at 25° C./20° C., 20° C./15°C. and 15° C./10° C., respectively. In each greenhouse, inoculatedplants were placed to observe the transition of disease symptoms.Control effect was judged with the average value from 6 repeated testsin the same manner as described in Test Example 6.

The test results are as follows.

                  TABLE 1                                                         ______________________________________                                        The Control Effect of a Composition comprising                                Xanthomonas strain P-482 and Imazosulfuron (Shibatait)                        Imazosulfuron                                                                              P-482                                                            (%)          0 CFU/ml    10.sup.8 CFU/ml                                      ______________________________________                                        (1) 25° C./20° C. (day/night), evaluation after 2 weeks         0            Control effect 0                                                                          Control effect 4.4                                   0.01         Control effect 0                                                                          Control effect 5.5                                   0.05         Control effect 0                                                                          Control effect 6.0                                   ______________________________________                                        (2) 20° C./15° C. (day/night), evaluation after 2 weeks         0            Control effect 0                                                                          Control effect 2.3                                   0.01         Control effect 0                                                                          Control effect 2.4                                   0.05         Control effect 0                                                                          Control effect 5.3                                   (3) 15° C./10° C. (day/night), evaluation after 3 weeks         0            Control effect 0                                                                          Control effect 0                                     0.01         Control effect 0                                                                          Control effect 0                                     0.05         Control effect 0                                                                          Control effect 3.5                                   ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        The Control Effect of a Composition comprising                                Xanthomonas strain P-484 and Flazasulfuron (Shibagen)                         Flazasulfuron                                                                             P-482                                                             (ppm)       0 CFU/ml      10.sup.8 CFU/ml                                     ______________________________________                                        (1) 25° C./20° C. (day/night), evaluation after 2 weeks         0           Control effect 0                                                                            Control effect 4.3                                  0.5         Control effect 0                                                                            Control effect 6.0                                  5           Control effect 0                                                                            Control effect 6.0                                  50            Control effect 2.2                                                                        Control effect 6.0                                  (2) 20° C./15° C. (day/night), evaluation after 2 weeks         0           Control effect 0                                                                            Control effect 2.1                                  0.5         Control effect 0                                                                            Control effect 2.9                                  5           Control effect 0                                                                            Control effect 4.9                                  50            Control effect 1.9                                                                        Control effect 5.4                                  ______________________________________                                    

As Tables 1- (1), -(2) and -(3) show, the mixed composition of thepresent invention comprising Xanthomonas strain P-482 and 0.05%imazosulfuron exhibits a synergistic increase in herbicidal activity ina relatively wide temperature range of from 10° C. to 25° C., comparedto the cases of the single application of either component.

As Tables 2-(1) and -(2) show, the mixed composition of the presentinvention comprising Xanthomonas strain P-484 and 5 ppm flazasulfuronexhibits a synergistic increase in herbicidal activity in a temperaturerange of from 15° C. to 25° C., compared to the cases of the singleapplication of either component.

USE EXAMPLE A Liquid Formulation comprising P-482 or P-484 andImazosulfuron

At the time of mixing, a P-482 or P-484 culture was adjusted to give acell density of 10⁸ CFU/ml and Shibatait was adjusted to give aconcentration of 5 μl/ml (0.05% by weight as imazosulfuron). To theresultant mixture, 10 μl (0.01% by weight) of a surfactant, Silwet L-77,was added. Then, the mixture was diluted with distilled water to give a100 ml liquid formulation. As test plants, there were used annualbluegrass (3 weeks from the sowing, which had been grown under the sameconditions as described in Test Example 2), Korean lawn grass andbentgrass (both of which had been grown in a pot for about one year andthen made turf). For each of the plants, inoculation was repeated 6times using 6 Jiffy Pots (5 cm x 5 cm x 5 cm). Control effect was judgedin the same manner as described in Test Example 5, taking the meanvalue. Before testing, annual bluegrass, Korean lawn grass and bentgrasswere cut to a height of 2 cm with an electric mower. 3 ml of the aboveliquid formulation was spray-inoculated to each plant with a sprayer. Asa control, plants were spray-inoculated with the equal amount ofdistilled water. The inoculated plants were placed in greenhouseswherein the temperature was set at 20° C./15° C. or 25° C./20° C.(day/night). Control effect was judged 2 weeks later. The results ofthis test are shown in Tables 3 and 4.

                  TABLE 3                                                         ______________________________________                                        The Effect of a Liquid Formulation comprising P-482                                          Annual Korean lawn                                                                              Bent-                                                       bluegrass                                                                            grass      grass                                        ______________________________________                                        (1) 20°C./15 °C. (day/night) , evaluation after 2 weeks         Control  Control effect                                                                            0        0        0                                      Liquid   Control effect                                                                            4.2      0        0                                      formulation                                                                   (2) 25° C./20° C. (day/night), evaluation after 2 weeks         Control  Control effect                                                                            0        0        0                                      Liquid   Control effect                                                                            6.0      0        0                                      formulation                                                                   ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        The Effect of a Liquid Formulation comprising P-484                                               Annual    Korean lawn                                                                            Bent-                                                      bluegrass grass    grass                                  ______________________________________                                        (1) 20° C./15° C. (day/night), evaluation after 2 weeks         Control  Control effect                                                                           0         0        0                                      Liquid   Control effect                                                                           4.3       0        0                                      formulation                                                                   (2) 25° C./20° C. (day/night), evaluation after 2 weeks         Control  Control effect                                                                           0         0        0                                      Liquid   Control effect                                                                           6.0       0        0.                                     formulation                                                                   ______________________________________                                    

From the above results, it is clear that both liquid formulationsrespectively comprising P-482 and P-484 can kill annual bluegrasscompletely without giving any damage to bentgrass or Korean lawn grass.

The herbicidal composition of the present invention shows excellentherbicidal activity against annual bluegrass, and it also showsherbicidal activity against broad-leaved weeds. Therefore, thecomposition of the present invention is extremely useful as a herbicidefor use at turf locations such as golf courses.

What is claimed is:
 1. A process for the control of annual bluegrass,which comprises applying to annual bluegrass a herbicidal compositioncomprising a microorganism having an ability to control annual bluegrassand belonging to Xanthomonas campestris, and a sulfonylurea compound. 2.The process according to claim 1, wherein said microorganism belongingto the species Xanthomonas campestris is Xanthomonas campestris P-482 orXanthomonas campestris P-484.
 3. The process according to claim 1,wherein said sulfonylurea compound is imazosulfuron, flazasulfuron,pyrazosulfuron-ethyl or bensulfuron-methyl.
 4. The process according toclaim 1, wherein said herbicidal composition is a liquid formulationwherein the microorganism concentration is 10¹ -10¹¹ CFU/ml.
 5. Theprocess according to claim 4, wherein the microorganism concentration ismore than 10³ CFU/ml.
 6. The process according to claim 1, wherein theconcentration of sulfonylurea is 0.001-0.1 weight percent.
 7. Theprocess according to claim 1, wherein the annual bluegrass to becontrolled is Poa annua.
 8. A process for controlling annual bluegrass,which comprises applying to annual bluegrass an effective amount of aherbicidal composition comprising a microorganism having an ability tocontrol annual bluegrass and having all of the identifyingcharacteristics of Xanthomonas campestris, and a sulfonylurea compound.9. A herbicidal composition for controlling annual bluegrass, whichcomprises herbicidally effective amounts of Xanthomonas campestrishaving the ability to control annual bluegrass and a sulfonylureacompound.
 10. The composition according to claim 9, wherein saidmicroorganism belonging to the species Xanthomonas campestris isXanthomonas campestris P-482 or Xanthomonas campestris P-484.
 11. Thecomposition according to claim 9, wherein said sulfonylurea compound isimazosulfuron, flazasulfuron, pyrazosulfuron-ethyl orbensulfuron-methyl.